POLY(RIBOADENYLIC ACID) IN
mRNA
Racker, E. (1951), J . Biol. Chem. 190, 685. Rose, Z . B., and Racker, E. (1962), J . Bid. Chem. 237,3279. Schwyzer, R., and Hurlimann, C. (1954), Helu. Chim. Acta 37,155. Simon, E. J., and Shemin, D. (1953), J . Amer. Chem. SOC.75, 2520. Stadtman, E. R. (1952), JBiol. Chem. 196,535. Stern, J. R., and Drummond, G. I. (1961), J. Bid. Chem. 236,
2892. Uotila, L. (1973), Biochemistry 12,3938, Vignais, P. V., and Zdbin, I. (1958), Biochim. Biophys. Acta 29,263. Vince, R., and Wadd, W. B. (1969), Biochem. Biophys. Res. Commun. 35,593. Webb, J. L. (1966), Enzyme and Metabolic Inhibitors, Vol. 11, NewYork,N. Y . ,AcademicPress,p751.
Characterization of Poly(riboadeny1ic acid) Segments in L-Cell Messenger Ribonucleic Acidt Joseph J. Eiden and J. L. Nichols*
The polyadenylate segments in L-cell mRNA have been characterized by the use of two-dimensional “fingerprinting” techniques. Digestion of poly(A)-containing mRNA with pancreatic ribonuclease and TI ribonuclease in high salt buffer releases the intact poly(A) segments ; these are readily separable from the (A-)nCp, (A-)nGp, and (A-)nUp components which arise by digestion from the remainder of the molecule. Digestion of the same mRNA preparations with both enzymes, but in low salt buffer, results in the hydrolysis of the poly(A) segments to oligonucleotides with chain
ABSTRACT:
P
oly(riboadeny1ic acid) [poly(A)] segments of up to 250 nucleotides in length have been reported to be covalently linked to heterogeneous nuclear RNA, eukaryotic messenger R N A (Lim and Canellakis, 1970; Darnell et al., 1971b; Lee et al., 1971; Edmonds et al.. 1971), and viral RNA (Kates, 1970; Lai and Duesberg, 1972). The functional role of untranslated regions in eukaryotic mRNA such as the poly(A) tracts is uncertain at this time. However, there is evidence to suggest that poly(A) may be necessary for processing and transport of mRNA from the nucleus to the cytoplasm (Darnell et al., 1971a). The recent demonstration that poly(A) tracts located at the 3 ’ terminus are longer in newly synthesized mRNA (Greenberg and Perry 1972a; Sheiness and Darnell, 1973) indicates that this region of the R N A may also serve to protect the informational part of the molecule from nucleolytic destruction during its life span in the cell. Further, the interaction of a heterogeneous population of mRNA molecules with ribosomes during translation or possibly with proteins during transport from the nucleus to the cytoplasm (Henshaw, 1968; Samarina et al., 1968; Perry and Kelley, 1968) would require invariable regions in mRNA. Regions of the R N A which could be involved in RNA-protein interactions are likely to be in the untranslated regions at the chain termini, such as the poly(A) segments. Evidence for the involvement of mRNA poly(A)
t From the Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710. Receiced June 4, 1973. This study was supported by Grant AI 10361 from the U. S. Public Health Service.
lengths 2 15; these oligo(adeny1ic acid) components are also largely separable from the (A-)nCp, (A-)nGp, and (A-)nUp components. Fractionation of the alkali hydrolysis products of the oligoadenylates or of poly(A) released by digestion in high salt buffer did not reveal the presence of guanylic, cytidylic, or uridylic acid. This suggests that poly(A) segments in L-cell mRNA are homopolymers of adenylic acid. Using the same methods, no poly(A) could be detected in L-cell ribosomal RNA.
segments in mRNA-protein interactions has been described (Kwan and Brawerman, 1972; Blobel, 1973). In order to gain some insight into the structure and posttranscriptional mechanism of the addition of poly(A) to mRNA, the isolation and nucleotide sequence analysis of the poly(A) segments in L-cell mRNA have been studied. The present report suggests that poly(A) tracts in chain lengths up to about 250 residues consist of only adenylic acid residues. No regions of the mRNA adjacent to the poly(A) segment are released with the poly(A) by nuclease digestion in high salt buffer. Materials and Methods Cells. Mouse L-cells were grown in Eagles minimum essential medium (Joklik-modified, Grand Island Biological Co.), containing 5 fetal calf serum. Labeling of Cells. L-Cells (1-2 X 108) in 30-ml cultures were grown for 3 to 4 hr at 37” in the presence of [2,8-3H]adenosine (150 pCi/ml) or carrier-free 3 2 P 0 4(175 pCi/ml). Labeling of cells in the presence of 32P04was done in phosphate-free medium. In some experiments labeling of cells was carried out after an initial 30-min incubation in the presence of actinomycin D (0.05 pg/ml, Calbiochem) or in the presence of actinomycin D (0.05 pg/ml) and ethidium bromide (1 pg/ml, Calbiochem). Cell Fractionation and Isolation of mRNA Containing Poly(A) Segments. Breakage of cells and preparation of postmitochondrial supernatants were accomplished by procedures described previously (Penman et al., 1963; Vesco and Penman, B I O C H E M I S T R Y , VOL.
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1 : Affinity chromatography on Sepharose-poly(U) columns of polysomal RNA isolated from cultures grown in the presence ( a ) and absence (b) of actinomycin D. The arrows indicate the point of addition of buffer (0.1 M NaC1-0.01 M Tris-HCI (pH 7.4) (1) and water (2). FIGCRE
1969). Polysomes were isolated by centrifuging the postmitochondrial supernatants for 2.5 hr through 40 % sucrose in RSBl at 40,000 rpm in a SW 50 rotor. Alternatively, polysomes were isolated from the interphase between a 60% sucrose cushion (in RSB) and a 5520% sucrose gradient (Lindberg and Persson, 1972). RNA was isolated from polysomes by the method of Lee et ul. (1971). The aqueous phase from the phenol deproteinization step was precipitated by the addition of three volumes of 95% ethanol and washed twice by resuspension and centrifugation in cold 95% ethanol (Nichols et al., 1972). The R N A samples were placed briefly under vacuum to remove ethanol and stored until use at -20". RNA containing poly(A) was isolated from the polysomal R N A preparations by affinity chromatography on Sepharose 4B-poly(uridylic acid) columns. The preparation of Sepharose-poly(U) has been described (Lindberg et a]., 1972). R N A was dissolved in 0.5 ml of NETS' buffer and applied to a column of Sepharose-poly(U) which had been washed with 50 ml of NETS buffer. The microcolumn contained 2 ml of a Sepharose-poly(U) slurry in a glass wool-stoppered Pasteur pipet. Unbound R N A was washed through the column with 10 mi of NETS buffer. Fractionsof 2 ml werecollected throughout. The column was then washed with 4 ml of buffer (0.1 M NaC1-0.01 hi Tris-HC1 (pH 7.4)) in order to remove sodium dodecyl sulfate and EDTA which were found to interfere with subsequent enzymic digestions and fractionations if they were present in samples containing small amounts of RNA. The R N A which remained bound to the column was then eluted with distilled water. No further R N A could be removed from the column by the use of a formamide buffer (Lindberg et ul.. 1972). The radioactivity in each fraction was determined by counting a 10-pl aliquot in scintillation fluid (Nichols et ul.. 1973). R N A was recovered by ethanol precipitation and washed twice with cold 95 % ethanol. Detection, Isolation, and Purification of PoI,,(A) Sequences. I Abbreviations used are: (A-)iiCp, (A-)nCp, and (A-)nUp, oligonuclcotides containing from 1 to 5 adenylic acid residues with a 3'-terminal cytidylic acid, guanylic acid, and uridylic acid residue, respectively; RSB, 0.01 M NaC1-0.01 \I Tris-HC1 (pH 7.2)-0.0015 h t MgCI?; KETS, 0.1 2.1 NaCI-0.01 M EDTA-0.01 h~ Tris-HCI ( p H 7.4)-0.2% sodium dxiecyl sulfate.
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32P-Labeled poly(A) segments were isolated by digestion with both T1ribonuclease (0.2 unit; Sankyo Co.) and pancreatic ribonuclease (0.2 pg; Worthington Biochemicals) in 100 p1 of high salt buffer (0.2 hi NaC1-0.01 M Tris-HC1 (pH 7.4)-0.01 M EDTA) for 1 hr at 37". The digest was then made 1 % in sodium dodecyl sulfate and 50 p g of carrier Escherichiu coli tRNA was added. The R N A was precipitated with ethanol and subjected to electrophoresis in 5 % polyacrylamide gels 1973). The gels were cut into 1 -mm sliccs and (Nichols et d.. the poly(A) peak was detected by Cerenkov radiation using a Beckman scintillation counter. The poly(A) material was extracted from the gel by methods described previously (Nichols, 1970). The presence of poly(A) segments in KNA was also determined by digestion of the R N A in 10 pl of high salt buffer containing 0.02 in it of T1 ribonuclease and 0.02 p g of pancreatic ribonuclease, for 15 min at 37". In this procedure the digestion products were fractionated by electrophoresis and homochromatography (Brownlee and Sanger, 1969). The thin layer plates used in the homochromatographic step hcrc made with a mixture of DEAE-cellulose and cellulose (Machcry, Nagel & Co.) in the proportions 1 :7.5. The "homomix" consisted of a 3 % partially hydrolyzed solution of R N A in 7 M urea. Material was eluted from the thin layer plates according to the procedure of Brownlee and Sanger (1969). Digestion of "P-labeled R N A with both Ti ribonuclease and pancreatic ribonuclease in low salt b u l k (0.01 M Tris-HCI (pH 7.4)-0.001 hi EDTA) was performed according to published procedures (Sanger et id.. 1965; Brownlee et d.. 1968). The digestion products were fractionated by electrophoresis and homochrotmtography as described above. Sucrose Densitj, Grudient Centrifirgu~ion.RNA samples i n 1 in1 of buffer (0.01 hi EDTA~-O.Olhi NaCI-0.01 XI Tris-HC1 (pH 7.5)) were layered over linear 30--60% sucrox gradients (in the same butkr) and centrifuged in an SW 25 rotor at 6" for 16 hr. Fractions were collected from the bottom of the tube and assayed for C1,CCOOH-precipitable counts. Isolation o f L-cell Virus LOM. Molecirlur Weiglit R N A . The 4s and 7 s R N A components of purified L-cell virus were isolated as described previously (Nichols et d..1973). 'IHLabeled 4S and 7 s components were detected in polyacrylamide gels following electrophoresis by treating each 1 -nim slice with Protosol (New England Nuclear) prior to counting in a toluene-based scintillation fluid. Alkuli Hjdro1j.si.c ctf RN.4 unil P o l j , ( A ) .R N A samples were hydrolyzed in 0.7 N NaOH for 18 hr at 37'. 'The resulting nucleoside monophosphates were resolved by paper electrophoresis at p H 3.5 (Sanger et ul.. 1965). Results Fractionation of L-cell polysomal R N A on Sepharose-poly(U) columns results in the separation of those ribonucleates containing covalently bound poly(A) regions from those which do not contain extensive poly(A) regions. Figure 1 illustrates the separation of these two R N A classes. The material which is not bound to the column is largely ribosomal R N A since incubation of the cells in low levels of actinomycin D (Figure la) prior to the addition of R2POi results in a much decreased incorporation of radioactivity into this fraction, whereas the R N A bound to the column shows a much smaller decrease in the level of incorporated radioactivity. Actinomycin D in the concentrations employed in this experiment (0.05 pg>ml)has been shown to preferentially suppress ribosomal R N A synthesis (Perry, 1963). When
POLY(RIBOADENYLIC
ACID) IN
mRNA
TABLE I: Composition of S e p h a r m P o l y ( U ) Column
Frac-
tions.'
PI
z
Nucleotide'
I
d rn
I. Unbound
0
25.1 23.1 34.0 17.8
UP CP GP AP 11. Water Eluent
UP CP GP Av
a
29.2 18.9 24.4 27.5
a The nucleoside 2' (3') monophosphates were resolved by paper electrophoresis at p H 3.5 (see Materials and Methods). The position of the nucleotides was determined by autoradiography and each nucleotide area was excised and counted in a toluene-based scintillation fluid (Nichols et a/., 1973). The values represent the mean of two independent determinations and are not corrected for C + U deamination (5 %).
cells were preincubated with both ethidium bromide and actinomycin D, the results were identical with those where just actinomycin D was employed (as in Figure la). This eliminates the possibility of mitochondrial R N A contamination in the preparations (Zylber et a/.,1969). Sucrose density gradient fractionation of the two classes of R N A derived from cultures labeled in the absence of actinomycin D and separated by Sepharose-poly(U) chromatography (illustrated in Figure Ib) is shown in Figure 2. Messenger R N A which binds to the column is quite heterogeneous with a peak intermediate between the 28s and 18s ribosomal R N A components of the material which does not bind to the column. The nucleotide compositions of the Sepharose-poly(U) bound and unbound RNAs are quite different; the mRNA (bound) contains a higher proportion of adenylic acid compared t o the unbound fraction (Table I). The values for the nucleotide composition of rRNA (unbound) are similar to those previously reported by other workers (Lane and Tamaoki, 1967; Faras and Erikson, 1969). The presence of poly(A) segments in mRNA eluted from Sepharose-poly(U) columns could be demonstrated by digestion of the RNA with TI and pancreatic ribonuclease in high salt buffer (see Materials and Methods), followed by fractionation of the digestion products using electrophoresis and homochromatography (Figure 3). The (A-)nGp, (A-)nCp, and (A-)nUp' components are well separated from the poly(A) which, because of its larger size, remains at the origin following the second-dimensional homochromatographic step. No large poly(A) segments could be detected if the same procedure was performed with unbound rRNA from Sepharose-poly(U) columns (data not shown). The poly(A) segments in the mRNA accounted for &lo% of the total 31P radioactivity when assayed by C l C C O O H precipitation or Sepharose-poly(U) chromatography following digestion in high salt buffer. It has previously been demonstrated that phosphodiester linkages involving adenylic acid residues become susceptible to the action of pancreatic ribonuclease under conditions of low ionic strength or suitably high concentrations of the
m
N
Froclion Number Sucrose density gradient profile of R N A fractionated by Sepharose-poly(U) chromatography. Fractions 1 and 2 (unbound RNA) and 8 and 9 (bound RNA) from the experiment illustrated in Figure lb were pooled separately and the ethanol-precipitated RNA samples were redissolved and r u n in parallel gradients (see Materials and Methods). CICCOOH-precipitable counts of the bound ( 0 )and unbound (0)RNA preparations in the gradient are plotted together. F ~ G V R E2:
enzyme (Lane and Butler, 1959; Beers, 1960). Thus, if digestion with both TI and pancreatic ribonuclease takes place in low salt buffer, the poly(A) segments in the R N A become susceptible to enzymic digestion resulting in oligoadenylates of varying sizes (Figure 4a). The oligoadenylates move in a unique position between the (A-)nCp and (A-)nGp components. Increasing the time of nuclease digestion results in the appearance of shorter oligoadenylates (data not shown) without detectably affecting the proportions of the (A-)nCp, (A-)nGp, and (A-)nUp components. Hydrolysis of the oligoadenylates with alkali showed that they contained only adenylic acid. Figure 4b shows the separation of products resulting from digestion with T, and pancreatic ribonuclease in low salt buffer of Sepharose-poly(U) unbound rRNA. In this case, no oligoadenylates are evident, demonstrating that the
3: Autoradiograph of the electrophoretic-homochromatographic separation of a T, and pancreatic ribonuclease digest of mRNA performed in high salt buffer:( I ) direction ofelectrophoresis in the first dimension; ( 2 ) direction of homochromatography in the second dimension.
FIGURE
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Fraction Number 5: Polyacrylamide gel electrophoresis of polysarnal RNA digested with T, and pancreatic ribonuclease in high salt buffer. Direction of electrophoresis is from left to right in relation to the figure: :~PP-labeled poly(A) ( 0 ) and tritiated 7s (chain length 280) and 4s (chain lensth 80) rihonucleates from L-cell virus (A). FIUURE
FIGURE 4: Autoradiographs of the electrophoretic-homochromatographic separations of T, and pancreatic ribonuclease digests of RNA performed in low salt buffer: (a) mRNA; (h) rRNA; (1) direction of electrophoresis in the first dimension; (2) direction of homochromatography in the second dimension
Sepharose-poly(U) column is selectively binding those species of R N A which contain large poly(A) segments. Poly(A) segments liberated by digestion of polysomal RNA in high salt buffer with TI and pancreatic ribonuclease and purified by electrophoresis on polyacrylamide gels show a heterogeneous profile with chain lengths up to about 250 nucleotides in length (Figure 5 ) . After polyacrylamide gel electrophoresis of nuclease digested 3*P-labeledpolysomal RNA, the poly(A) was extracted from the gel and subjected to digestion with TI and pancreatic ribonuclease in either high or low salt buffer. Figure 6a shows the fractionation of the isolated poly(A) after digestion in high salt buffer. No radioactive components other than the poly(A) (which remains on the origin) could be detected. Digestion in low salt buffer (Figure 6b) releases oligoadenylates of different chain lengths. N o material in the (A-)nGp, (A-)nCp, or (A-)nUp regions could be detected. Analysis of each of these oligoadenylates by alkali hydrolysis demonstrated the presence of only adenylic acid. Because of the methods of analysis employed, it could not be determined which if any of those components had cyclic end groups. With oligoadenylates greater than 10 nucleotides in chain length, the recovery of radioactive material from the thin layer plate for subsequent analyses was decreased, and it was difficult,
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therefore, to rule out the possibility that these components might contain guanylic, cytidylic, or uridylic acid at their 3’ ends. However, when the time of digestion was increased, smaller oligonucleotides (
